Functional Analysis Of PKS5 And Its Interacting Partner Chaperon DnaJ In ABA Signaling

PKS5 (SOS2-like Protein Kinase 5) belongs to PKS family in Arabidopsis, which have 25 members. PKS5 encodes a protein of 435 amino acids. PKS5 plays an important role in protecting plants from suffering high pH stress by phosphorylating and inhibiting the plasma membrane H+-ATPase activity. Expression of PKS5 was up-regulated by ABA, NaCl, mannitol, glucose, dehydration or drought, suggesting that PKS5 may involve in multiple biological processes in Arabidopsis.In order to study the function of PKS5, one T-DNA insertion line and six Tilling (Targeted induced local lesion) mutants of PKS5 were obtained from ABRC(Arabidopsis Research Center). The homozygous lines were tested under different treatments of abiotic stresses or hormones. Two of Tilling mutants, pks5-6 and pks5-7 were found hypersensitive to ABA only at germination stage. However the T-DNA knockout line was not more sensitive to ABA than wild type. In pks5-6 mutant, a transition of c-t causes the substitution of serine317 to leucine. In pks5-7, the c-t transition causes Alanine168 changed to Valine. PKS5-7 mutation lies in the kinase activation loop of PKS5 protein, and PKS5-6 mutation locates at FISL motif. These two domains are conservative in PKS family.To investigate the possible reasons why only pks5-6 and pks5-7 showed the ABA sensitive phenotype, PKS5 and all of the PKS5 mutant proteins were tested for their kinase activities. Our data indicated that the kinase activity of PKS5-6 and PKS5-7 increased 5-8 times comparing that of PKS5.In order to identify what protein will interact with PKS5 in ABA signaling, I screened a cDNA expression library with a yeast two-hybrid system using PKS5 as bait. Totally, 23 PIPs (PKS5 interacting protein) were acquired. which involved in ROS signaling pathway, or photosynthesis pathway, or 26S protein degradation complex, or molecular co-chaperon mechanism or other unknown expression proteins.Phosphorylation assays were performed to investigate whether these PIPs would be the substrates of PKS5. Results showed that PKS5 could phosphorylate PIP133 and truncated PIP55 (1-181aa) in vitro. PIP145, which encodes a DnaJ-like protein, showed strong interaction with PKS5. More Y2H assays were conducted to define the interacting domains of PKS5 and DnaJ using the truncated proteins. The C terminal of PKS5 strongly interacted with the DnaJ C terminal, the FISL motif and PPI domain of PKS5 are essential for their interaction, and the whole DnaJ C terminal was also important.To confirm whether the two proteins can interact in vivo, Co-IP assays were done using a transient protoplast expression system. The results were assured. Furthermore DnaJ inhibited the kinase activity of PKS5.Both knockout alleles of DnaJ (dnaj13 and dnaj14) have the similar phenotype as pks5-6 and pks5-7 mutants, they are very sensitive to ABA at the same period. The GUS staining showed that DnaJ and PKS5 highly expressed in the roots of young seedlings, which provides the premise for their synergetic functions.Consistent with our biochemical results, phenotype of double mutant of dnaj13 pks5 was contrary to that of dnaj13 pks5-7 and dnaj13 pks5-6. dnaj13 pks5 was not more sensitive to ABA than wild type, while dnaj13 pks5-7 and dnaj13 pks5-6 are much more sensitive to ABA than their parents dnaj13, pks5-7 and pks5-6.This study has confirmed that the kinase activity of PKS5 is determinative for ABA phenotype at germination stage, thus identified novel functions of protein kinase of PKS5 and its interacting partner DnaJ in ABA signaling.